The Role And Mechanism Of Down-regulation Of UHRF1 In Oxidative Damage Of Endothelial Cells In Diabetes

Objective Oxidative stress has been demonstrated to play a crucial role in inducing vascular endothelium dysfunction and diabetic vasculopathy.The specific mechanism of impaired endothelial cell function in diabetes has not been fully elucidated.Kelch-like ECH-associated protein 1（KEAP1）/nuclear factor E2-related factor 2（NRF2）/antioxidant response element signaling pathway is one of the most important intracellular defense mechanisms against oxidative stress.Impaired NRF2 transcriptional activity in diabetes aggravates oxidative damage,and long-term hyperglycemia can lead to epigenetic changes,affecting chromatin configuration and expression of related genes.Ubiquitin like,containing PHD and RING finger domains 1（UHRF1）is an important epigenetic regulator that recognizes DNA methylation and histone modification and is widely involved in maintaining DNA stability and regulation of cell cycle,apoptosis,and proliferation.UHRF1 is closely related to the occurrence and development of various malignant diseases,including tumor angiogenesis.Therefore,we hypothesized that UHRF1 might play a critical role in regulating endothelial cell function.This study aimed to investigate the relationship between UHRF1 and oxidative stress-induced endothelial cell dysfunction in diabetes.We tried to clarify the effects of in vitro and in vivo glucolipotoxicity on the expression of UHRF1 protein levels,and also to evaluate the effects of UHRF1 down-regulation on impaired endothelial cell function.The epigenetic mechanism whereby UHRF1 regulated KEAP1/NRF2signaling pathway in endothelial cells was also investigated.Methods（1）Endothelial colony-forming cells（ECFCs）were isolated and expanded ex vivo from the peripheral blood of patients with type 2 diabetes mellitus（T2DM）.Western blot was used to detect the protein levels of UHRF1,KEAP1,and NRF2.The protein expression level of UHRF1 was also analyzed in endothelial cells incubated with high glucose,palmitic acid（PAL）,or hydrogen peroxide（H₂O₂）.（2）After transient silencing of UHRF1,the migration ability,apoptosis,and reactive oxygen species（ROS）production of human umbilical vein endothelial cell（HUVEC）and ECFC were evaluated.Western blot was used to measure the protein levels of KEAP1,NRF2,phosphorylation of NRF2 at Ser40（p-NRF2）,heme oxygenase-1（HO-1）,NAD（P）H:quinone oxidoreductase 1,and cleaved cysteinyl aspartate specific proteinase-3（cleaved caspase-3,a marker for measuring apoptosis）after UHRF1knockdown in HUVECs.In addition,the effects of UHRF1overexpression on migration capacity of T2DM ECFCs and cleaved caspase-3,p-NRF2,and HO-1 protein levels in HUVECs exposed to a fixed high concentration of PAL were confirmed using scratch assay and western blot assay,respectively.（3）In an endothelial cell injury model of H₂O₂-induced oxidative damage,UHRF1 protein expression level in ECFCs was observed after the antioxidant intervention using glutathione（GSH）.The cleaved caspase-3 protein expression level,cellular apoptosis rate,and ROS production of HUVECs were detected under combined treatment of GSH and PAL after silencing UHRF1.（4）The simultaneous knockdown of UHRF1 and KEAP1 model in HUVECs was used to observe cellular migration ability by scratch assay and to measure p-NRF2 and HO-1 protein levels.UHRF1 protein expression level and ROS production were measured after NRF2 knockdown.（5）Mechanistically,activity of the promoter of KEAP1 gene was detected by dual-luciferase reporter assay system after either down-regulation or up-regulation of UHRF1.After UHRF1 silencing in HUVECs,methylation-specific PCR and bisulfite sequencing PCR were used to qualitatively and quantitatively analyze the methylation status of Cp G island in KEAP1 promoter region,respectively.Chromatin immunoprecipitation（Ch IP）was used to detect the enrichment of histone H3 lysine 27 acetylation（H3K27Ac）within KEAP1 promoter region after UHRF1 silencing or up-regulation in HUVECs.Results（1）Compared with non-diabetic patients,the protein levels of UHRF1 and NRF2 in peripheral blood-derived ECFCs of patients with T2DM were decreased,while the KEAP1 protein level was increased.UHRF1 protein levels were also significantly decreased in endothelial cells treated with high glucose,PAL,or H₂O₂ in vitro.（2）The migration ability of HUVECs and normal ECFCs was impaired after UHRF1down-regulation,accompanied with increased cell apoptosis rate,protein level of cleaved caspase-3,and ROS production.Overexpression of UHRF1 significantly improved the migration ability of diabetic ECFCs and decreased the protein level of cleaved caspase-3 in PAL-treated HUVECs.（3）In the normal ECFCs treated with H₂O₂,GSH co-treatment improved cell morphology and increased UHRF1 protein level;knockdown of UHRF1 attenuated the protective effect of GSH on PAL-induced HUVEC apoptosis.（4）After silencing UHRF1 in HUVECs,the p-NRF2/HO-1 pathway was inhibited.Furthermore,the inhibitory effect of UHRF1 silencing on HUVEC migration ability and protein expression levels of p-NRF2 and HO-1 was improved by additional depletion of KEAP1.In HUVECs incubated with PAL,the impaired p-NRF2/HO-1 axis was rescued by UHRF1 up-regulation.（5）KEAP1promoter activity was increased after UHRF1 knockdown in HUVECs and decreased after UHRF1 up-regulation.The bisulfite sequencing PCR results showed that total methylation levels of the selected Cp G site exhibited no significant differences between UHRF1 silencing group and negative control group in HUVECs.Ch IP-q PCR analysis showed that UHRF1 silencing resulted in increased enrichment of H3K27Ac at the promoter region of KEAP1 gene,and UHRF1 overexpression decreased the H3K27Ac level.（6）NRF2 knockdown in turn inhibited UHRF1protein expression,enhanced ROS production,and promoted oxidative stress-induced injury in HUVECs.Conclusions Oxidative stress can mediate oxidative damage of diabetic endothelial cells by downregulating the expression of UHRF1,which enhances ROS production by regulating KEAP1/p-NRF2 pathway through histone acetylation and might form a self-perpetuating feedback loop with KEAP1/p-NRF2 to further promote oxidative stress-induced apoptosis of endothelial cells in diabetes.